UV / VISIBLE SPECTROSCOPY

MASH’HOOD MAHMODD KHAN SHAHIDMUHAMMAD UZAIR AMIR JUTTMUHAMMAD SAIFULLAH KHAN

SPECTROSCOPY

Study of spectrum formed by interaction of matter with radiant energy.

Spectroscopy is the branch of science that involves the study of interaction of electromagnetic radiations with atoms or molecules.

It is of two types; Emission spectroscopy Absorption spectroscopy

Emission spectroscopy

It is the measure of emitted light and spectroscopic analysis of this emitted light gives the emission spectrum.

An emission spectrum thus obtained gives the information about the light source under study.

This phenomenon is primarily caused by excitation of atoms by the thermal or electrical means, absorbed energy causes electron in the ground state to be promoted to the state of higher energy where they are short lived and will return to the ground state by emitting the radiations.

But there may also be phosphorescence in some instances.

Example: Flame photometry Flourimetry

Absorption spectroscopy

It is the measure of energy absorbed by an excited atom or molecule and the spectroscopic analysis of the energy gives the absorption spectrum.

It also represents the energy absorbed relative to the energy of given frequency of electromagnetic radiation.

Example: IR UV/VISIBLE NMR

UV/VISIBLE SPECTROSCOPY:- Uv-vis spectroscopy is also

known as electronic spectroscopy. In which the amount of light absorbed at each wavelength of Uv and visible regions of electromagnetic spectrum is measured. This absorption of electromagnetic radiations by the molecules leads to molecular excitation.

Electromagnetic radiations

Principle

The absorption of energy by ground state atom in the gaseous state forms the basis of atomic absorption spectroscopy.

Spectrum is a graph of intensity of absorbed or emitted radiation by sample verses frequency (ν) or wavelength (λ).

A phenomenon of interaction of molecules with ultraviolet and visible lights.

Absorption of photon results in electronic transition of a molecule, and electrons are promoted from ground state to higher electronic states.

Principle is based upon the electronic transitions: σσ → → σσ* transition* transitionππ → → ππ* transition* transitionn → n → σσ* transition* transitionn → n → ππ* transition* transitionσσ → → ππ* transition* transitionππ → → σσ* transition* transition

Electronic transition

σ → → σ* transition* transition

σ electron from orbital is excited to corresponding anti-bonding orbital σ*.

Methane (CH4) has C-H bond only

n → n → σσ* transition* transition

Saturated compounds containing atoms with lone pair of electrons like O, N, S and halogens are capable of n → σ* transition..

ππ → → ππ* transition* transition

π electron in a bonding orbital is excited to corresponding anti-bonding orbital π*.

Compounds containing multiple bonds like alkenes, alkynes, carbonyl, nitriles, aromatic compounds, etc undergo π → π* transitions.

n → n → ππ* transition* transition

An electron from non-bonding orbital is promoted to anti-bonding π* orbital.

Compounds containing double bond involving hetero atoms (C=O, C≡N, N=O) undergo such transitions.

instrumentation

Following are the general components of atomic absorption spectrometer:Sources of Radiation (UV and Visible)Wavelength selector (Monochromator)Cuvette (sample and refrence container)Detectors Signal processor (amplifier) and read-out device.

Radiation source

UV:Deutrium and hydrogen lamps.The electrical excitation of deuterium or hydrogen at low pressure produces a continuous UV spectrum.Range: 160-375nmPrecaution:Quartz windows and quartz cuvette must be used b/c glass absorb radiation of wavelength less then 350nm.

Radiation source

Visible source:Tungston filament lampRange: 350-2500nmThe lifetime of a tungsten/halogen lamp is approximately double that of an ordinary tungsten filament lamp.

Wavelength selector

There are following components in every monochromators namely:Entrance slitCollimating lensDispersing device (prism of grating)Focusing lensExit slit

cuvette

The containers for the sample and reference solution must be transparent to the radiation which will pass through them. Quartz or fused silica cuvettes are required for spectroscopy in the UV region. These cells are also transparent in the visible region. Silicate glasses can be used for the manufacture of cuvettes for use between 350 and 2000 nm.

Detector Photomutiplier tube Linear photodiode array Charged coupled devices

http://en.wikipedia.org/wiki/File:Schematic_of_UV-_visible_spectrophotometer.png

applications Detection of impurities Structure elucidation of organic compound Quantitative analysis Qualitative analysis Dissociation constants of acids and base Chemical kinetics Quantitative analysis of pharmaceutical analysis Molecular weight determination. As HPLC detector.

Detection of Impurities  UV absorption spectroscopy is one of the best methods for

determination of impurities in organic molecules. Additional peaks can be observed due to impurities in the sample and it can be compared with that of standard raw material. By also measuring the absorbance at specific wavelength, the impurities can be detected.Benzene appears as a common impurity in cyclohexane. Its presence can be easily detected by its absorption at 255 nm.

The bands due to impurities are very intense. Saturated compounds show little absorption while unsaturated

show strong absorption bands.

Structure elucidation of organic compounds

UV spectroscopy is useful in the structure elucidation of organic molecules, the presence or absence of unsaturation, the presence of hetero atoms.From the location of peaks and combination of peaks, it can be concluded that whether the compound is saturated or unsaturated, hetero atoms are present or not etc.

Quantitative analysis UV absorption spectroscopy can be used for the quantitative

determination of compounds that absorb UV radiation. This determination is generally based on Beer’s Law;

which states that Absorption is directly proportional to Conc. of sample in the solution.

For this, we produce calibration curve for different standard concentrations of the sample solution. Then unknown sample concentration can be determined by comparing the values in standard graph.

Qualitative analysis UVabsorption spectroscopy can characterize those types of

compounds which absorbs UV radiation. Identification is done by comparing the absorption spectrum with the spectra of known compounds.UV absorption spectroscopy is generally used for characterizing aromatic compounds and aromatic olefins.

Dissociation constants of acids and bases.

PH = PKa + log [A-] / [HA]From the above equation, the PKa value can be calculated if the ratio of [A-] / [HA] is known at a particular PH. and the ratio of [A-] / [HA] can be determined spectrophotometrically from the graph plotted between absorbance and wavelength at different PH values.

Chemical kinetics Kinetics of reaction can also be studied using UV spectroscopy.

The UV radiation is passed through the reaction cell and the absorbance changes can be observed.

 Quantitative analysis of pharmaceutical

substances  Many drugs are either in the form of raw material or in the form

of formulation. They can be assayed by making a suitable solution of the drug in a solvent and measuring the absorbance at specific wavelength.Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at the wavelength 284 nm.

Molecular weight determination Molecular weights of compounds can be measured

spectrophotometrically by preparing the suitable derivatives of these compounds.For example, if we want to determine the molecular weight of amine then it is converted in to amine picrate. Then known concentration of amine picrate is dissolved in a litre of solution and its optical density is measured at λmax 380 nm. After this the concentration of the solution in gm moles per litre can be calculated by using the following formula.

"c" can be calculated using above equation, the weight "w" of amine picrate is known. From "c"and "w", molecular weight of amine picrate can be calculated. And the molecular weight of picrate can be calculated using the molecular weight of amine picrate.

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